Chlamydia trachomatis is a major cause of pelvic inflammatory disease and secondary infertility. During infection, mononuclear and polymorphonuclear phagocytes elaborate a variety of substances such as cytokines and reactive oxygen species which may be responsible for the long-term sequelae of infection. The goal of this proposal is to define the molecular basis of phagocyte activation by CT constituents in the hopes of defining rational targets for intervention in infected patients. We have approached the problem of phagocyte activation by C. trachomatis by defining four areas for initial investigation: the chlamydial lipopolysaccharide, the presence of cytokines in chlamydial infected tissues, bioactive CT derived proteins and the interaction of CT with leukocyte beta2 integrins (CD11a-c/CD18). We will begin by characterizing the efficacy and potency of chlamydial LPS as a stimulus for monocytes (cytokine and prostaglandin release) and PMN (superoxide production, enhanced CR3 expression) and comparing CT LPS to standard LPS preparations and gonococcal LPS. We will ask if CT LPS interacts with the same leukocyte LPS receptors as enteric endotoxins and if exogenous sexual steroids regulate the induction of cytokines in monocytes. In collaboration with Dr. K. Takayama (Univ. of WI), we will define the structure of the CT lipid A using HPLC purification of lipid A combined with FAB-mass, laser desorption and NMR spectrometry. The second approach to defining the molecular basis of phagocyte activation will be to investigate protein constituents of CT. We will begin by producing recombinant CT major outer membrane protein (MOMP) and a recombinant chlamydial heat shock protein (hsp60) which have been implicated as important molecules in immune activation. The ability of these molecules to activate phagocytes independently of LPS will be assessed by the use of the previously mentioned LPS inhibitors and cells which have been rendered specifically tolerant to LPS. The presence of additional active CT proteins will be further assessed using a recently described "monocyte Western blotting" approach. In addition, we will provide collaborators with DNA probes to help define where and when cytokines are produced in infected tissues in situ. Finally, we will define the interactions of chlamydia with CD11/CD18 leukocyte integrins, which bind the outer membranes of other gram-negative bacteria. We will test the binding of C. trachomatis and its purified membrane constituents to phagocytes employing monoclonal antibodies and a unique CD18-deficient mutant monocyte cell line. Finally, we will investigate how ligation of CD11/CD18 by C. trachomatis influences cellular activation as assessed by cytokine production. An understanding of how C. trachomatis interacts with phagocytic leukocytes ultimately should result in the development of novel therapies designed to reduce the serious morbidity of this widely prevalent and serious infection.